Reconstruction of a human cornea by the self-assembly approach of tissue engineering using the three native cell types

[摘要] Purpose: The purpose of this study wasto produce and characterize human tissue-engineered corneasreconstructed using all three corneal cell types (epithelial, stromal,and endothelial cells) by the self-assembly approach. Methods: Fibroblasts cultured in mediumcontaining serum and ascorbic acid secreted their own extracellularmatrix and formed sheets that were superposed to reconstruct a stromaltissue. Endothelial and epithelial cells were seeded on each side ofthe reconstructed stroma. After culturing at the air-liquid interface,the engineered corneas were fixed for histology and transmissionelectron microscopy (TEM). Immunofluorescence labeling of epithelialkeratins, basement membrane components, Na+/K+-ATPaseα1,andcollagentype I was also performed. Results: Epithelial and endothelialcells adhered to the reconstructed stroma. After 10 days at theair-liquid interface, the corneal epithelial cells stratified (4 to 5cell layers) and differentiated into well defined basal and wing cellsthat also expressed Na+/K+-ATPase α1 protein,keratin 3/12, and basic keratins. Basal epithelial cells from thereconstructed epithelium formed many hemidesmosomes and secreted a welldefined basement membrane rich in laminin V and collagen VII.Endothelial cells formed a monolayer of tightly-packed cells and alsoexpressed the function related protein Na+/K+-ATPaseα1.Conclusions: This study demonstrates thefeasibility of producing a complete tissue-engineered human cornea,similar to native corneas, using untransformed fibroblasts, epithelialand endothelial cells, without the need for exogenous biomaterial.

[学科分类] 生物化学/生物物理